Antibodies and methods for the detection of cell death

ABSTRACT

Disclosed herein are antibodies having binding specificity to the amino acid sequences Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1) and Thr Val Glu Val Asp (SEQ ID NO:14), and methods of detecting cell death in a sample, comprising contacting the sample with a first antibody specific for a C-terminal amino acid sequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1) or Thr Val Glu Val Asp (SEQ ID NO:14) of a CK18 protein fragment having a C-terminal amino acid sequence of Val Glu Val Asp (SEQ ID NO:2) and a second antibody that specifically binds an epitope that is present in both full-length CK18 and the CK18 protein fragment, and that does not overlap with SEQ ID NO:1 or SEQ ID NO:14, under conditions such that the CK18 protein fragment present in the sample specifically binds to the first antibody and the second antibody, wherein one of the antibodies is bound to a solid support and the other antibody is bound to a detection moiety capable of producing a signal; optionally removing any unbound or excess material; and detecting the signal from the detection moiety, wherein the signal is positively correlated with the presence of the CK18 protein fragment in the sample.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/127,644, filed Sep. 20, 2016, a national stage application ofInternational Application No. PCT/US2015/021678, filed internationallyon Mar. 20, 2015, which claims priority to U.S. Provisional PatentApplication Nos. 61/968,016, filed Mar. 20, 2014, and 61/987,668, filedMay 2, 2014. The contents of each of the above-references applicationsare hereby incorporated by reference in their entireties.

FIELD

The present disclosure relates to the detection of cell death. Inparticular, the present disclosure relates to antibodies and assaymethods used to detect cell death.

BACKGROUND

Apoptosis is the process of programmed cell death (PCD) that may occurin multicellular organisms. Biochemical events lead to characteristiccell changes (morphology) and death. These changes include blebbing,cell shrinkage, nuclear fragmentation, chromatin condensation, andchromosomal DNA fragmentation. In addition to its importance as abiological phenomenon, defective apoptotic processes have beenimplicated in an extensive variety of diseases. Excessive apoptosiscauses atrophy, whereas an insufficient amount results in uncontrolledcell proliferation, such as cancer.

Caspases are proteins that are highly conserved, cysteine-dependent,aspartate-specific proteases. There are two types of caspases: initiatorcaspases, caspase 2, 8, 9, and 10, and effector caspases, caspase 3, 6and 7. The activation of initiator caspases requires binding to specificoligomeric adaptor protein. Effector caspases are then activated bythese active initiator caspases through proteolytic cleavage. The activeeffector caspases then proteolytically degrade a host of intracellularproteins to carry out the cell death program.

Detection and measurement of apoptosis has been found to be useful indetecting the presence or severity of diseases. Cytokeratins (CK) areknown to be useful markers for many different diagnostic methods. Forexample, circulating fragments of cytokeratin 18 (also CK18, CK-18, K18,or K-18), a cytoskeletal marker of cell death, such as hepatocyte death,have been shown in several studies to indicate the transition frombenign fatty liver to nonalcoholic steatohepatitis (NASH), with a riskof fibrosis, in patients with nonalcoholic fatty liver disease (NAFLD).

NAFLD is a spectrum of disorders characterized by hepatic steatosis,which may be benign (nonalcoholic fatty liver or NAFL) or which mayprogress via inflammation and fibrosis to NASH followed by cirrhosis andliver failure. Liver biopsy, the standard diagnostic approach forNAFL/NASH, has limitations due to sampling site variability, cost andprocedure-related morbidity. Appropriate circulating biomarkers mayenable diagnosis, staging and monitoring of NAFL/NASH with fewerbiopsies.

Assays incorporating CK18 as a biomarker may be developed to monitor theseverity of such diseases by measuring apoptosis. CK18 is also usefulfor detecting and monitoring other liver diseases including hepatitis,biliary sclerosis, and poisoning. CK18 is also useful for quantifyingcell death in cancer and diseases that are part of the metabolicsyndrome, including degenerative diseases, such as cardiovascular andliver disease.

This disclosure covers antibodies and related assays and methods fordetecting cell death by measuring the presence of CK18 fragments, thatare typically present only when a cell is dying and after full-lengthCK18 is exposed to and cleaved by caspases.

SUMMARY

The disclosure relates generally to antibodies having bindingspecificity to the amino acid sequence Ala Ser Ser Gly Leu Thr Val GluVal Asp (SEQ ID NO:1). The disclosure also relates generally toantibodies having binding specificity to the amino acid sequence Thr ValGlu Val Asp (SEQ ID NO:14).

The disclosure relates generally to methods of producing an antibodythat binds selectively to a cytokeratin 18 (CK18) protein fragmenthaving a C-terminal amino acid sequence of Val Glu Val Asp (SEQ IDNO:2), comprising immunizing a mammal with an antigen having aC-terminal amino acid sequence of Ala Ser Ser Gly Leu Thr Val Glu ValAsp (SEQ ID NO:1) or Thr Val Glu Val Asp (SEQ ID NO:14) and isolatingthe antibody.

The disclosure relates generally to methods of producing an antibodythat binds selectively to a cytokeratin 18 (CK18) protein fragmenthaving a C-terminal amino acid sequence of Val Glu Val Asp (SEQ IDNO:2), comprising immunizing a mammal with an antigen having aC-terminal amino acid sequence of Ala Ser Ser Gly Leu Thr Val Glu ValAsp (SEQ ID NO:1) or Thr Val Glu Val Asp (SEQ ID NO:14) and generating acell line producing a monoclonal antibody.

The disclosure relates generally to methods of detecting cell deathand/or a cytokeratin 18 (CK18) protein fragment having a C-terminalamino acid sequence of Val Glu Val Asp (SEQ ID NO:2) in a sample,comprising the steps of contacting the sample with a first antibodyspecific for a C-terminal amino acid sequence Ala Ser Ser Gly Leu ThrVal Glu Val Asp (SEQ ID NO:1) of the CK18 protein fragment or for aC-terminal amino acid sequence Thr Val Glu Val Asp (SEQ ID NO:14) of theCK18 protein fragment and a second antibody that specifically binds anepitope that is present in both full-length CK18 and the CK18 proteinfragment, and that does not overlap with SEQ ID NO:1 or SEQ ID NO:14,under conditions such that the CK18 protein fragment present in thesample specifically binds to the first antibody and the second antibody,wherein one of the antibodies is bound to a solid support and the otherantibody is bound to a detection moiety capable of producing a signal;optionally removing any unbound or excess material; and detecting thesignal from the detection moiety, wherein the signal is positivelycorrelated with the presence of the CK18 protein fragment in the sample.

The disclosure relates generally to methods of determining theoccurrence of cell death in a sample, comprising the steps of combiningthe sample with a first antibody that has binding specificity to theamino acid sequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ IDNO:1) or to the amino acid sequence Thr Val Glu Val Asp (SEQ ID NO:14)and a second antibody that specifically binds an epitope that is presentin both full-length CK18 and a CK18 protein fragment having a C-terminalamino acid sequence of Val Glu Val Asp (SEQ ID NO:2), and that does notoverlap with SEQ ID NO:1 or SEQ ID NO:14, under conditions such that theCK18 protein fragment present in the sample specifically binds to thefirst antibody and the second antibody, wherein one of the antibodies isbound to a solid support and the other antibody is bound to a detectionmoiety; performing an immunological assay using the first antibody andsecond antibody; determining the presence in the sample of acaspase-cleaved cytokeratin 18 (CK18) protein fragment having aC-terminal amino acid sequence of Val Glu Val Asp (SEQ ID NO:2), whereinthe presence of the CK18 protein fragment is positively correlated tocell death. The determining step further comprising detecting a signalfrom the detection moiety attached to one of the antibodies, whereindetection of the signal is positively correlated with the presence ofthe CK18 protein fragment.

The disclosure generally relates to kits for detecting a cytokeratin 18(CK18) protein fragment having a C-terminal amino acid sequence of ValGlu Val Asp (SEQ ID NO:2) in a sample, the kit comprising a solidsupport; a detection moiety; a first antibody specific for a C-terminalamino acid sequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ IDNO:1) of the CK18 protein fragment; or for a C-terminal amino acidsequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1) of theCK18 protein fragment; a second antibody that specifically binds anepitope that is present in both full-length CK18 and the CK18 proteinfragment, and that does not overlap with SEQ ID NO:1 or SEQ ID NO:14;and instructions for performing the detection assay.

Apart from the subject matter discussed above, the present disclosureincludes a number of other exemplary features such as those explainedhereinafter. It is to be understood that both the foregoing descriptionand the following description are exemplary only.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a C-terminal amino acid sequence (²²⁹ASSGLTVEVD) of acytokeratin 18 (CK18) protein fragment.

SEQ ID NO:2 is the C-terminal amino acid sequence (²³⁵VEVD) of thecaspase-cleaved fragment of SEQ ID NO:3 and SEQ ID NO:4.

SEQ ID NO:3 is the amino acid sequence of full-length human cytokeratin18 (CK18) protein.

SEQ ID NO:4 is the amino acid sequence of a caspase-cleaved fragment(M1-D238) of cytokeratin 18 (CK18) protein, also referred to as“fragment B.”

SEQ ID NO:5 is the amino acid sequence of the variable region of theheavy chain of mAb 3C2.

SEQ ID NO:6 is the nucleotide sequence of the variable region of theheavy chain of mAb 3C2.

SEQ ID NO:7 is the amino acid sequence of the variable region of thelight chain of mAb 3C2.

SEQ ID NO:8 is the nucleotide sequence of the variable region of thelight chain of mAb 3C2.

SEQ ID NO:9 is the amino acid sequence ³⁹⁴DALD.

SEQ ID NO:10 is the amino acid sequence (²³⁹APKSQDLAKI) of a CK18peptide.

SEQ ID NO:11 is the amino acid sequence (²⁴⁰PKSQDLAKI) of a CK18peptide.

SEQ ID NO:12 is the amino acid sequence (²²⁹ASSGLTVEV) of a CK18peptide.

SEQ ID NO:13 is the amino acid sequence (³⁸⁸EDFNLGDALD) of a CK18peptide.

SEQ ID NO:14 is the C-terminal amino acid sequence (²³⁵TVEVD) of thecaspase-cleaved fragment of SEQ ID NO:3 and SEQ ID NO:4.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying table and figures are incorporated in, and constitute apart of this specification.

FIG. 1 is a schematic drawing of an assay format of an embodimentdisclosed herein;

FIG. 2 is a graphical illustration of hybridoma specificity tested forantibodies disclosed herein;

FIG. 3 is a photographic representation of a Western Blot Analysis witha commercially available monoclonal antibody (mAb) C-04;

FIG. 4 is a photographic representation of a Western Blot Analysis withmAb 3C2;

FIG. 5 is a graphical illustration of antibody epitope specificity forantibodies disclosed herein;

FIG. 6 is a graphical representation of the detection of a recombinantCK18 fragment B (rCK18fB) which serves as a representative standardcurve for the methods described in Example 5;

FIG. 7 is a graphical representation of the evaluation of normal, NAFLand NASH samples as described in Example 6;

FIG. 8 is a graphical representation of a curve prepared with rCK18fB,according to the methods disclosed herein; and

FIG. 9 is a graphical representation of a curve prepared with SK-BR3Apoptotic Supernatant according to the methods disclosed herein.

DETAILED DESCRIPTION Definitions

Unless specifically defined otherwise herein, all technical, scientific,and other terms used herein have the same meaning as commonly understoodby one of ordinary skill in the art of immunoassays and relatedsciences.

The term “detection moiety” as used herein, refers to a moiety that isdetectable and/or capable of producing a detectable signal, and can beattached, directly or indirectly, to various binding partners fordetection in an assay.

The term “solid support” as used herein, refers to any material that isinsoluble and/or has structural rigidity and resistance to changes ofshape or volume, and to which a binding partner, such as an antibody,can be immobilized or bound.

“N-terminus,” “N-terminal” or “amino terminus” refers to the amino acidresidues which are closest to the amino-terminus of a protein.

“C-terminus,” “C-terminal” or “carboxy terminus” refers to the aminoacid residues which are closest to the carboxy-terminus of a protein.

Antibodies

Human cytokeratin protein CK18 is a 430 amino acid protein (SEQ ID NO:3)which may be fragmented by the activity of the proteases caspase 3 andcaspase 6 during cell death or cellular apoptosis. This activitygenerates three protein fragments having the sequences M1-D238 (SEQ IDNO:4) (also referred to as “Fragment B”), A239-D397 and 5398-H430(sequences not provided). It is believed that detection of CK18 proteinfragments having the C-terminal sequence VEVD (or Val Glu Val Asp) (SEQID NO:2) may be used as an indication of cell death, which may be usefulin detection and diagnosis of degenerative diseases and cancer.Accordingly, one aspect of the present disclosure is directed to anantibody to a CK18 protein fragment having the C-terminal sequence ofSEQ ID NO:2.

Another aspect of the present disclosure is directed to an antibodywhich specifically binds to the amino acid sequence Ala Ser Ser Gly LeuThr Val Glu Val Asp (SEQ ID NO:1), or a functionally equivalentsequence. Another aspect of the present disclosure is directed to anantibody which specifically binds to the amino acid sequence Thr Val GluVal Asp (SEQ ID NO:14), or a functionally equivalent sequence. Thefunctionally equivalent sequences should include at least the sequenceVal Glu Val Asp (SEQ ID NO:2). A functionally equivalent sequence refersto a sequence that is homologous in function, comprising at least theC-terminal amino acid sequence Val Glu Val Asp (SEQ ID NO:2) havingreplacements of one or several amino acids in the above identified aminoacid sequences (SEQ ID NO:1 and SEQ ID NO:14) with other amino acids,other molecular forms of the amino acids, non-natural amino acids and/orderivatives, as long as the three-dimensional structure of the SEQ IDNO:1 or the SEQ ID NO:14 is mimicked.

In some embodiments, the antibody has binding specificity to the aminoacid sequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1), andhas a variable region of the heavy chain amino acid sequence of SEQ IDNO:5, and a variable region of the light chain amino acid sequence ofSEQ ID NO:7. In some embodiments, the antibody has binding specificityto the amino acid sequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQID NO:1), and has a variable region of the heavy chain nucleotidesequence of SEQ ID NO:6, and a variable region of the light chainnucleotide sequence of SEQ ID NO:8.

In some embodiments, the antibody has binding specificity to the aminoacid sequence Thr Val Glu Val Asp (SEQ ID NO:14), and has a variableregion of the heavy chain amino acid sequence of SEQ ID NO:5, and avariable region of the light chain amino acid sequence of SEQ ID NO:7.In some embodiments, the antibody has binding specificity to the aminoacid sequence Thr Val Glu Val Asp (SEQ ID NO:14), and has a variableregion of the heavy chain nucleotide sequence of SEQ ID NO:6, and avariable region of the light chain nucleotide sequence of SEQ ID NO:8.

The sequence Val Glu Val Asp (SEQ ID NO:2) is uncleaved within intact,full-length CK18 proteins such that full-length CK18 proteins do notreact with an antibody having binding specificity for the sequencesreferenced above (i.e., SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:14). Thisis significant for measuring cell death where, in such instances, theCK18 protein has been cleaved by caspases.

Another aspect of the present disclosure is directed to a secondantibody that binds to an epitope that is present in (1) full-lengthCK18; (2) a CK18 protein fragment having the C-terminal sequence VEVD(SEQ ID NO:2); or (3) both (1) and (2), where the epitope has adifferent amino acid sequence from SEQ ID NO:1 and SEQ ID NO:14, i.e.,little to no overlap with the amino acids in SEQ ID NO:1 and SEQ IDNO:14. The second antibody may be, for example, any suitable orcommercially available monoclonal antibody. The second antibody may beused to identify and confirm the presence of the CK18 protein fragmenthaving the C-terminal sequence VEVD (SEQ ID NO:2).

Antibody Development

Monoclonal antibodies may be developed to particular epitopes/antigensof the cytokeratin 18 protein and cytokeratin 18 protein fragments.These monoclonal antibodies may be used in Western blots, thedevelopment of ELISAs (enzyme-linked immunosorbent assays), as well asin the development of other assay formats includingelectrochemiluminescence (ECL)-based assays, for different forms of CK18that are found, for example, in body fluids, such as blood, plasma,serum, etc. Standard procedures known to one of skill in the art may beused to generate hybridomas, which may produce monoclonal antibodiesdirected against the protein or peptide of interest.

One aspect of the present disclosure is directed to methods ofdeveloping antibodies that bind selectively to a CK18 protein fragmenthaving a C-terminal amino acid sequence of Val Glu Val Asp (SEQ IDNO:2). In some embodiments, antibodies that bind selectively to a CK18protein fragment having a C-terminal amino acid sequence of Val Glu ValAsp (SEQ ID NO:2) may be developed by immunizing a mammal with anantigen having a C-terminal amino acid sequence of Ala Ser Ser Gly LeuThr Val Glu Val Asp (SEQ ID NO:1) or Thr Val Glu Val Asp (SEQ ID NO:14)and isolating the antibody.

In other embodiments, antibodies that bind selectively to a CK18 proteinfragment having a C-terminal amino acid sequence of Val Glu Val Asp (SEQID NO:2) may be developed by immunizing a mammal with an antigen havinga C-terminal amino acid sequence of Ala Ser Ser Gly Leu Thr Val Glu ValAsp (SEQ ID NO:1) or Thr Val Glu Val Asp (SEQ ID NO:14) and generating acell line producing a monoclonal antibody.

Assay Development and Methods

The antibodies disclosed herein may be used in various formats of assaysand methods that measure, quantify, and/or detect the presence of celldeath as well as CK18 protein fragments having the C-terminal sequenceof Val Glu Val Asp (SEQ ID NO:2). FIG. 1 is a schematic drawing of anassay format of an embodiment disclosed herein. The antibodies describedherein can be combined with a sample to perform the assays. The samplemay be a biological sample, such as tissue extracts, tissues used inimmunohistochemistry, or fluids. The fluid samples may be derived fromblood, plasma, serum, ascites fluid, saliva, sputum or urine.

The antibodies described herein may be linked or bound to variouscomponents or moieties in order to perform assay functions. For example,in some embodiments, the antibodies discussed herein may be bounddirectly through covalent or non-covalent attachment, or indirectly to asolid support or carrier. When bound indirectly, intermediate linkersmay be used to bind the components. Suitable intermediate linkersinclude, but are not limited to, an amino group or a carboxylate group,biotin, ligands, or other chemical bonds. Suitable solid supports orcarriers include, but are not limited to, glass surfaces (e.g., a glassslide or bead), plastic surfaces, metal surfaces, polystyrene surfaces(e.g., a bead or a plate), nitrocellulose surfaces, microparticles,nanoparticle surfaces, plates, wells, disposable ECL electrodes, andparamagnetic or magnetic beads that may be coated with avidin orstreptavidin or have other surface functionalities to promote bindingaffinity. In FIG. 1, a streptavidin-coated paramagnetic bead is depictedas bound to a biotin-linked anti-VEVD mAb.

In some embodiments, the antibodies discussed herein may be linked orbound, directly through covalent or non-covalent attachment, orindirectly, to a detection moiety. When bound indirectly, intermediatelinkers may be used as discussed herein. In some embodiments, thedetection moiety may be any detection moiety that corresponds to asuitable detection method. FIG. 1 depicts an anti-CK18 mAB bound to TAG,an ECL detection moiety used in ECL-based detection methods.

Suitable detection moieties include, but are not limited to,electrochemiluminescence labels or compounds, chemiluminescentcompounds, enzyme labels, fluorophores, chromogenic compounds,radiolabels, catalysts, colorimetric compounds or labels, labeledantibodies, a latex particle, a magnetic or paramagnetic particle, aradioactive element, fluorescent dyes, phosphorescent dyes, dyecrystals, gold particles, silver colloidal particles, selenium colloidalparticles, metal chelates, coenzymes, electro active groups,oligonucleotides or stable radicals. The metal chelate may be aruthenium, an osmium or a europium metal chelate. The detection methodmay include any known detection method including, but not limited to,chromogenic, radioisotopic, fluorescence, immunofluorescence,luminescence, bioluminescence, and electrochemiluminescence (ECL).

In some embodiments, the detection method may beelectrochemiluminescence (ECL). An electrochemiluminescent compound mayserve as the detection moiety that may be detected or quantified withinan ECL reaction chamber, such as in a flow cell, or on a disposableelectrode. The solid support may serve to hold the antibody bound to thedetection moiety near an ECL electrode in the ECL reaction chamberduring detection.

Electrochemiluminescence (ECL) is the process whereby a molecularspecies, such as an “ECL label,” luminesces upon the exposure of thatspecies to electrochemical energy in an appropriate surrounding chemicalenvironment. ECL is a rapid and sensitive bio-analytical detectiontechnique that is a regenerative process. Some of the advantagesachieved with ECL as a detection method in biological sample analysisinclude simpler, less expensive instrumentation; stable, nonhazardouslabels; and increased assay performance characteristics such as lowerdetection limits, higher signal to noise ratios, and lower backgroundlevels. Certain applications of ECL have been developed and reported inthe literature. U.S. Pat. Nos. 5,147,806, 5,068,808, 5,061,445,5,296,191, 5,247,243, 5,221,605, 5,238,808, 5,310,687, 5,714,089,6,165,729, 6,316,607, 6,808,939, 6,881,589, 6,881,536, and 7,553,448,the disclosures of which are incorporated herein by reference, detailcertain methods, apparatuses, chemical moieties, inventions, andassociated advantages of ECL.

Electrochemiluminescence signals are generated by a redox reactionbetween an electrochemiluminescent detection moiety, such as anECL-active label with a redox substrate that occurs at the surface of anelectrode. In certain embodiments, the ECL label is a ruthenium(Ru)-containing reagent. One example of a suitableelectrochemiluminescent label is Tris(bypyridine)ruthenium(II)([Ru(bipy)3]²⁺), also referred to as TAG (See, e.g., FIG. 1). In someembodiments, the redox substrate is tripropylamine (TPA).

In some embodiments, a magnet usually positioned below an electrode mayattract the paramagnetic beads, pulling down the Ru-labeled complex nearthe electrode. The Ru may then be oxidized. Oxidized tripropylamine(TPA) may react with the oxidized Ru, which then may emit a photon. Theredox reaction between Ru and the redox substrate tripropylamine (TPA)that occurs only in the electric field near the electrode may be aregenerative process during continued application of voltage, whichallows for an ECL signal that undergoes amplification over time. Becausephotons can only be generated near the electrode surface,electrochemiluminescence only occurs when the Ru is brought intoproximity with the electrode by the magnet, thereby reducing backgroundlevels. Nonspecific ECL is not triggered by any known naturalconstituents of biological samples; therefore, unlike chemiluminescence,which often displays background artifacts due to nonspecific triggeringof chemiluminescent detector molecules, ECL maintains reduced backgroundlevels.

In some embodiments, the solid support and/or the detection moiety maybe from a lyophilized composition that is rehydrated with the sample foruse in an assay. The lyophilized composition may contain standard and/orother necessary assay specific components of an assay, such as buffers,reagents, detergents, preservatives, salts, proteins, antibodies, etc.It is contemplated that the solid support and the detection moiety maybe lyophilized in separate compositions, and then rehydrated with thesample. It is also contemplated that the solid support and the detectionmoiety may be lyophilized in the same composition, and then rehydratedwith the sample. It is further contemplated that the lyophilizedcompositions disclosed herein may be rehydrated by one or more otherassay specific components discussed herein.

The antibodies of the present disclosure may be used in various assayformats, including, for example, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunoparticle assays (ELIPA) or ECL assays fordetecting cell death or the presence of CK18 fragments. In one aspect ofthe present disclosure, the assay method steps for detecting acytokeratin 18 (CK18) protein fragment having an amino acid sequence ofVal Glu Val Asp (SEQ ID NO:2) in a sample may include contacting thesample with a first antibody specific for a C-terminal amino acidsequence Ala Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1) of theCK18 protein fragment and a second antibody that specifically binds anepitope that is present in both full-length CK18 and the CK18 proteinfragment, and that does not overlap with SEQ ID NO:1, under conditionssuch that the CK18 protein fragment present in the sample specificallybinds to the first antibody and the second antibody, wherein one of theantibodies is bound to a solid support and the other antibody is boundto a detection moiety capable of producing a signal; optionally removingany unbound or excess material, such as the sample, assay reagents,antibodies or protein fragments; and detecting the signal from thedetection moiety, wherein the signal is positively correlated with thepresence of the CK18 protein fragment in the sample.

In another aspect of the present disclosure, the assay method steps fordetecting a cytokeratin 18 protein fragment having an amino acidsequence of Val Glu Val Asp (SEQ ID NO:2) in a sample may includecontacting the sample with a first antibody specific for a C-terminalamino acid sequence Thr Val Glu Val Asp (SEQ ID NO:14) of the CK18protein fragment and a second antibody that specifically binds anepitope that is present in both full-length CK18 and the CK18 proteinfragment, and that does not overlap with SEQ ID NO:14, under conditionssuch that the CK18 protein fragment present in the sample specificallybinds to the first antibody and the second antibody, wherein one of theantibodies is bound to a solid support and the other antibody is boundto a detection moiety capable of producing a signal; optionally removingany unbound or excess material, such as the sample, assay reagents,antibodies or protein fragments; and detecting the signal from thedetection moiety, wherein the signal is positively correlated with thepresence of the CK18 protein fragment in the sample.

In some embodiments, the first antibody may be bound to a solid supportand the second antibody may be bound to a detection moiety. In otherembodiments, the first antibody may be bound to a detection moiety andthe second antibody may be bound to a solid support.

In one aspect of the present disclosure, the assay method steps fordetermining the occurrence of cell death in a sample may includecombining the sample with a first antibody that has binding specificityto the C-terminal amino acid sequence Ala Ser Ser Gly Leu Thr Val GluVal Asp (SEQ ID NO:1) and a second antibody that specifically binds anepitope that is present in both full-length CK18 and a CK18 proteinfragment having a C-terminal amino acid sequence of Val Glu Val Asp (SEQID NO:2), and that does not overlap with SEQ ID NO:1, under conditionssuch that the CK18 protein fragment present in the sample specificallybinds to the first antibody and the second antibody, wherein one of theantibodies is bound to a solid support and the other antibody is boundto a detection moiety; performing an immunological assay using the firstantibody and the second antibody; determining the presence in the sampleof a caspase-cleaved cytokeratin 18 protein fragment having a C-terminalamino acid sequence of Val Glu Val Asp (SEQ ID NO:2), wherein thepresence of the CK18 protein fragment is positively correlated to celldeath.

In another aspect of the present disclosure, the assay method steps fordetermining the occurrence of cell death in a sample may includecombining the sample with a first antibody that has binding specificityto the C-terminal amino acid sequence Thr Val Glu Val Asp (SEQ ID NO:14)and a second antibody that specifically binds an epitope that is presentin both full-length CK18 and a CK18 protein fragment having a C-terminalamino acid sequence of Val Glu Val Asp (SEQ ID NO:2), and that does notoverlap with SEQ ID NO:14, under conditions such that the CK18 proteinfragment present in the sample specifically binds to the first antibodyand the second antibody, wherein one of the antibodies is bound to asolid support and the other antibody is bound to a detection moiety;performing an immunological assay using the first antibody and thesecond antibody; determining the presence in the sample of acaspase-cleaved cytokeratin 18 protein fragment having a C-terminalamino acid sequence of Val Glu Val Asp (SEQ ID NO:2), wherein thepresence of the CK18 protein fragment is positively correlated to celldeath.

In some embodiments, the first antibody may be bound to a solid supportand the second antibody may be bound to a detection moiety. In otherembodiments, the first antibody may be bound to a detection moiety andthe second antibody may be bound to a solid support.

In some embodiments, the determining step may also include detecting asignal from the detection moiety attached to one of the antibodies,wherein detection of the signal is positively correlated with thepresence of the CK18 protein fragment.

In some embodiments, the change in the amount of cell death may bedetermined. The determined change in the amount of cell death or cellapoptosis may be used in the diagnosis of diseases with involvement ofapoptosis. The determined change in the amount of cell death may also beused in the monitoring of an effect of therapy of diseases withinvolvement of apoptosis. Such diseases may include cancer ordegenerative diseases, such as cardiovascular and liver disease.

It is contemplated that the steps of the methods of the presentdisclosure do not have to be completed in the order provided herein, andmay be performed in different orders, where, for example, the antibodiesmay be added to or contacted with the sample sequentially or at the sametime. It is contemplated that the first antibody may be added first,followed by the addition of the second antibody, or the second antibodymay be added first, followed by the addition of the first antibody.

If the antibodies are added sequentially, it is contemplated that thesample may be incubated for a period of time after the addition of eachantibody and before the next method step(s). Additionally, the samplemay be incubated for a period of time before the washing step andremoval of any unbound or excess materials. It is further contemplatedthat additional washing steps to remove materials during the assay maybe performed at additional times during the method, such as after theaddition of each antibody, after the addition of both antibodiestogether and/or before the detecting step.

Kits

Another aspect of the present disclosure is directed to kits forperforming the methods described herein. For example, a kit may be usedfor detecting cell death in a sample and/or a cytokeratin 18 (CK18)protein fragment having a C-terminal amino acid sequence of Val Glu ValAsp (SEQ ID NO:2) in a sample. Materials to be included in the kit mayvary depending on the ultimate purpose. As such, the kits may includeone or more components that are used in the methods.

The kits disclosed herein may include one or more of the followingcomponents: an antibody specific for a C-terminal amino acid sequenceAla Ser Ser Gly Leu Thr Val Glu Val Asp (SEQ ID NO:1) of the CK18protein fragment; an antibody specific for a C-terminal amino acidsequence Thr Val Glu Val Asp (SEQ ID NO:14) of the CK18 proteinfragment; an antibody to a CK18 protein fragment having the C-terminalsequence VEVD (or Val Glu Val Asp) (SEQ ID NO:2); an antibody thatspecifically binds an epitope that is present in both full-length CK18and a CK18 protein fragment having the C-terminal sequence VEVD (SEQ IDNO:2), and that does not overlap with SEQ ID NO:1 or SEQ ID NO:14; adetection moiety; a solid support; assay reagents; buffers; standardsand instructions for performing the methods disclosed herein, as well asother components and elements of the methods described herein. Thestandards can be additional chemical reagents or data (empirical) inprinted or electronic form necessary for the calibration needed forperformance of the assay.

The kit may include a vehicle within which some or all of the componentsare stored, carried or used for processing, such as a disposablecartridge. The kit may also include the use of an analyzer instrument,such as an ECL analyzer, and include instructions for use and relatedinstrument components, such as cartridges used with the analyzerinstrument.

The present disclosure can be better understood by reference to theexamples included herein, which illustrate but do not limit the presentteachings described herein. It is to be understood that both thedescriptions disclosed herein and the following examples are merelyillustrative and intended to be non-limiting.

EXAMPLES

The following examples are intended to be non-restrictive andexplanatory only.

Example 1—3C2 Antibody Selection

Caspases 3 and 6 generated three CK18 protein fragments having thesequences M1-D238 (SEQ ID NO:4), A239-D397 and 5398-H430. A monoclonalantibody specific for the carboxy terminus of human caspase-cleavedcytokeratin CK18 protein fragment having amino acids M1-D238 (SEQ IDNO:4) was generated by immunizing mice with a short peptide sequence of²²⁹ASSGLTVEVD (SEQ ID NO:1) (designated as peptide P1 for the Examples).Mouse sera were screened for a positive response to peptide P1.Splenocytes from mice with a positive response were fused with myelomacells and cell culture fluid from the resulting hybridomas screenedagainst peptide P1. Supernatant from hybridoma cells producing antibodywith a positive response to peptide P1 were further screened by anelectrochemiluminescence (ECL) assay against biotin conjugated peptides²³⁹APKSQDLAKI (SEQ ID NO:10), ²⁴⁰PKSQDLAKI (SEQ ID NO:11), ²²⁹ASSGLTVEVD(SEQ ID NO:1) and ²²⁹ASSGLTVEV (SEQ ID NO:12), which were each bound tostreptavidin coated paramagnetic beads. Peptides ²²⁹ASSGLTVEVD (SEQ IDNO:1) and ²²⁹ASSGLTVEV (SEQ ID NO:12) represent the amino acid sequencesA229-D238 and A229-V237 of cytokeratin 18 protein while peptides²³⁹APKSQDLAKI (SEQ ID NO:10) and ²⁴⁰PKSQDLAKI (SEQ ID NO:11) representthe amino acid sequences A239-I248 and K240-I248 of cytokeratin 18.

FIG. 2 provides a graphical illustration of the specificity of hybridomacell lines producing antibodies with positive response to peptide P1.Cell lines 2A4, 3C2, 14B6, 12E4, 12C2, 20E3 and 15D9 were found to bespecific for the sequence ²²⁹ASSGLTVEVD (SEQ ID NO:1) and negative forpeptide sequences ²²⁹ASSGLTVEV (SEQ ID NO:12), ²³⁹APKSQDLAKI (SEQ IDNO:10) and ²⁴⁰PKSQDLAKI (SEQ ID NO:11) (See, e.g., FIG. 2).

Example 2—Characterization of Monoclonal Antibody 3C2

Monoclonal antibodies 3C2 and C-04 were characterized by Western blotanalysis against full length recombinant cytokeratin 18 protein (rCK18)and a cell extract of apoptotic SK-BR3 cells (human breast cancer cellline induced to become apoptotic). FIG. 3 is a photographic reproductionof a Western Blot Analysis using a commercially available monoclonalantibody C-04 (Sigma-Aldrich®, SAB4700411-100UG) (Lane 1, markerproteins; Lane 2, blank; Lane3, 1 μg recombinant full length CK18protein (rCK18); Lane 4, 12 μL of SK-BR3 cell apoptotic supernatant;Lane 5, 12 μL of SK-BR3 cell supernatant). Proteins were run in a 14%Tris/Glycine gel. The concentrations for the test material were 1 μg ofrCK18 and 12 μL of SK-BR3 apoptotic supernatant and SK-BR3 cell lysate.The proteins were run at 150V constant until the dye front reached theend of the gel. The proteins were then transferred to nitrocellulose.The nitrocellulose was blocked and then probed with the C-04 mAb at 1μg/mL for 60 minutes. The membrane was then washed three times. Themembrane was then probed with a goat anti-mouse IgG ALK for 30 minutes.The membrane was washed three times and developed using a WesternBreeze®kit from Invitrogen™ (Cat #WB7104). Monoclonal antibody C-04, specificfor human cytokeratin 18 protein, detected rCK18 (See FIG. 3, lane 3),full length CK18 from a cell extract of SK-BR-3 cells (See FIG. 3, Lane5), human CK18 protein fragment M1-D238 (SEQ ID NO:4) (See FIG. 3, lane4) and two additional smaller fragments.

FIG. 4 is a photographic reproduction of a Western Blot Analysis usingthe mAb 3C2 (Lane 1, marker proteins; Lane 2, blank; Lane 3, 1 μgrecombinant full length CK18 protein (rCK18); Lane 4, 12 μL of SK-BR3apoptotic cell supernatant; Lane 5, 12 μL of SK-BR3 cell supernatant).Proteins were run in a 14% Tris/Glycine gel. The concentrations for thetest material were 1 μg of rCK18 and 12 μL of SK-BR-3 apoptoticsupernatant and SK-BR-3 Cell Lysate. The proteins were run at 150 Vconstant until the dye front reached the end of the gel. The proteinswere then transferred to nitrocellulose. The nitrocellulose was blockedand probed with the 3C2 mAb at 1 μg/mL for 60 minutes. The membrane waswashed three times and probed with a goat anti-mouse IgG ALK for 30minutes. The membrane was washed three times and developed with using aWesternBreeze® kit from Invitrogen™ (Cat #WB7104). The results indicatedthat mAb 3C2 did not bind to full length rCK18 protein (See FIG. 4, lane3) or to full length recombinant CK18 protein (See FIG. 4, lane 5) anddid detect the presence of human CK18 protein fragment M1-D238 (SEQ IDNO:4) as well as smaller protein fragments which may be degradationproducts of this fragment (See FIG. 4, lane 4).

The specificity of monoclonal antibody 3C2 was also assessed in animmunoassay against the peptides ²²⁹ASSGLTVEVD (SEQ ID NO:1),²²⁹ASSGLTVEV (SEQ ID NO:12) and ³⁸⁸EDFNLGDALD (SEQ ID NO:13) (eachconjugated with biotin at their amino-terminus), which were added to a96-well microplate coated with NeutrAvidin® by Invitrogen™. Humancytokeratin 18 protein fragment A239-D397 was also produced by theaction of caspase 3 during cellular apoptosis and ends in the sequence³⁹⁴DALD (SEQ ID NO:9). These peptides were incubated with mAb 3C2, M30antibody (Enzo Life Sciences®, ALX-804-590) or other unrelated mAbs.

FIG. 5 provides a graphical representation of the antibody specificityfor the antibodies discussed above. The results summarized in the graphof FIG. 5, indicated that mAb 3C2 specifically bound to the peptideending in ²³⁵VEVD (SEQ ID NO:2) and did not bind to the peptides endingin ²³⁵VEV or ³⁹⁴DALD (SEQ ID NO:9), while the M30 antibody bound to thepeptide ending in ³⁹⁴DALD (SEQ ID NO:9) but did not bind to the peptidesending in ²³⁵VEVD (SEQ ID NO:2) or ²³⁵VEV.

Example 3—Amino Acid and Nucleotide Sequences of the Variable Regions ofthe Heavy and Light Chains of mAb 3C2

Sequences were obtained using standard procedures by LakePharma ofBelmont, Calif. Total RNA was extracted from hybridoma cells. RACE(Rapid Amplification of cDNA Ends) was performed to amplify DNA forvariable heavy (V_(H)) and variable light (V_(L)) chains. Positiveclones were identified by gel electrophoresis. Positive DNA was clonedand sequenced. The DNA was analyzed and the amino acid sequences forV_(H) and V_(L) were presented.

The amino acid sequence of the variable region of the heavy chain of mAb3C2 was determined to be: DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQAPEKGLEWVAY INGGSTIIYY ADTVKGRFTV SRDNPKNTLF LQMTSLRSDD TAMYFCVRRTPTAAGGAMDY WGQGTSVTVS S (SEQ ID NO:5).

The nucleotide sequence coding for the variable region of the heavychain of mAb 3C2 was determined to be: gatgtgcagc tggtggagtc tgggggaggcttagtgcagc ctggagggtc ccggaaactc tcctgtgcag cctctggatt cactttcagtagttttggaa tgcactgggt tcgtcaggct ccagagaagg ggctggaatg ggtcgcatacattaatggtg gcagtaccat catctactat gcagacacag tgaagggccg attcaccgtctccagagaca atcccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgacgacacggccatgt atttttgtgt aagaaggacc cctacggctg cggggggggc tatggactattggggtcaag gaacctcagt caccgtctcc tca (SEQ ID NO:6).

The amino acid sequence of the variable region of the light chain of mAb3C2 was determined to be: NIMVTQSPSS LAVSAGEKVT MTCKSSQSLF YSSNQKNYLAWFQQKPGQSP KLLIYWASTR ESGVADRFTG SGYGTDFTLT ISSVQAEDLA VYYCQQYLSSWTFGGGTKVE IK (SEQ ID NO:7).

The nucleotide sequence coding for the variable region of the lightchain of mAb 3C2 was determined to be: aacattatgg tgacacagtc gccatcatctctggctgtgt ctgcaggaga aaaggtcact atgacctgta agtccagtca aagtcttttctacagttcaa accagaagaa ctacttggcc tggttccagc agaaaccagg gcagtctcctaaactgctga tctactgggc atccactagg gagtctggtg tcgctgatcg cttcacaggcagtggatatg ggacagattt tactctaacc atcagcagtg tacaggccga agacctggcagtttattact gtcaacaata cctctcgtct tggacgttcg gtggaggtac caaggtggaaatcaaac (SEQ ID NO:8).

Example 4—Specificity for Caspase-Cleaved Cytokeratins

Assay specificity was examined using full length recombinant CK18(Fitzgerald Cat. #30R-2135), CK14 (Fitzgerald Cat. #30R-2140), CK18(Fitzgerald Cat. #30R-2139), CK19 (Fitzgerald Cat. #30R-2136), and CK20(Fitzgerald Cat. #30R-2142). The cytokeratins were used at 150 ng/mL offragmented material spiked into normal human serum and tested in theCK18 assay.

To prepare fragmented material the full length cytokeratins were treatedwith Caspase-3 (Sigma-Aldrich®, Cat. #C1224) by adding 10 μL of 1 mg/mLstock cytokeratin to 50 μL of buffer (50 mM Hepes, 100 mM NaCl, 5% (w/v)glycerol, 0.5 mM EDTA, 0.05% w/v) CHAPs and 5 mM DTT, pH 7.3) andtreated with 1 μL of recombinant Caspase-3 (0.2 μg/mL) for 2 hours at37° C. After caspase-3 treatment material was diluted to 150 ng/mL innormal serum and evaluated.

The capture mAb 3C2 was biotin-conjugated and pre-bound to streptavidincoated paramagnetic beads (See, e.g., FIG. 1). The detector mAb C-04 wasTAG-conjugated. Fifty microliters of calibrators (0-100,000 U/L),controls (High, Medium, Low), normal human serum spiked with 150 ng/mLfragmented cytokeratins and normal human serum spiked with 150 ng/mLfull length cytokeratins were combined with 50 μL of a master mixconsisting of equal volumes of capture and detector reagents in a 96well microplate and incubated for 60 minutes at room temperature withshaking. The plate was washed and read on an ECL analyzer.

The results show that the assay only produced signal with thecaspase-3-cleaved (treated) CK-18 and not with the other fourcaspase-3-cleaved (treated) cytokeratins or the full-length (untreatedwith caspase-3) cytokeratins. Table 1 contains the determinedconcentration results for the full-length (untreated) cytokeratins andcleaved (caspase-3-treated) cytokeratins. The data shows that the mAb3C2 does not bind to full-length or caspase-3-cleaved cytokeratins 8,14, 19 and 20.

TABLE 1 Cleaved Full Length (Caspase- (Untreated) 3-Treated) SpecificityMean Mean Samples (U/L) (U/L) CK-8 ND ND CK-14 ND ND CK-18 ND 176 CK-19ND ND CK-20 ND ND ND = Not Detected

Example 5—Human CK18 Protein Fragment B (rCK18fB) Assay

A recombinant human CK18 protein fragment B (rCK18fB) was prepared usingstandard procedures. An immunoassay consisted of incubating 50 μLrCK18fB (0, 206, 617, 1852, 5557, 16667, 50,000 and 100,000 U/L) spikedinto normal human serum with 50 μL of reaction mix consisting ofbiotinylated mAb 3C2/streptavidin coated paramagnetic beads andanti-CK18 TAG labeled antibody C-04 for 60 minutes at room temperaturewith shaking. The bead complex was washed twice, suspended in buffer andread on an ECL analyzer. The results shown in FIG. 6, a representativestandard curve, indicate rCK18fB is detectable over a range of 0-100,000U/L (1 U/L=1000 pg=0.037 pM).

Example 6—Detection of Human Cytokeratin 18 Protein Fragments in NAFLand NASH

Cytokeratin 18 protein fragments having C-terminus VEVD²³⁸ (SEQ ID NO:2)present in human plasma samples with known NAFL and NASH status werequantified using a rCK18fB standard curve, for example, as shown in FIG.6. Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorderscharacterized by hepatic steatosis, which may be benign (NAFL) or whichmay progress via inflammation and fibrosis to nonalcoholicsteatohepatitis (NASH) and then to cirrhosis and liver failure. Liverbiopsy is the standard diagnostic approach for NAFL/NASH. However it haslimitations due to sampling site variability, cost and procedure-relatedmorbidity. Appropriate NAFLD-specific circulating biomarkers may enablediagnosis, staging and monitoring of NAFL/NASH with fewer biopsies.Commercial human serum samples (50 normal, 68 NAFL and 29 NASH) wereobtained from ProMedDx and BioreclamationlVT. The samples and rCK18fBprotein (50 μL) were incubated as described in Example 5. The results(See, e.g., FIG. 7) indicated that the normal, NAFL and NASH sampleswere each readily quantified by the assay method (62, 288, and 755 U/L,respectively).

Example 7—Human CK18 Protein Fragment B Capture and Detector AntibodyOrientation

The sensitivity of the immunoassay was assessed with respect to the useof mAb C-04 in conjunction with the VEVD (SEQ ID NO:2) specific mAb 3C2by evaluating the orientation of the antibodies used to either captureor detect cytokeratin 18 protein fragments ending in the sequence VEVD(SEQ ID NO:2) including: a) Capture 3C2 with Detector C-04; and b)Capture C-04 with Detector 3C2.

The capture mAb was biotin-conjugated and pre-bound to streptavidincoated paramagnetic beads. The detector mAb was TAG-conjugated. Theworking concentration of pre-bound capture mAb beads was 0.2 mg/mL andthe working concentration of TAG-conjugated detector mAb was 5 μg/m L.The antibody orientation was evaluated with recombinant CK-18 fragment Bprotein (rCK18fB) and with a cell lysate from SK-BR-3 cells treated with50 μM of cisplatin for 48 hours to induce apoptosis. Samples (50 μL) ofrCK18fB (0-150,000 pg/mL in normal serum) and SK-BR-3 cell lysate(diluted from 1:2 to 1:128 by serial dilution in Antibody Diluent) werecombined with 50 μL of a master mix consisting of equal volumes ofcapture and detector reagents (representing each of the four capture anddetector mAb pairs) in a 96 well microplate and incubated for 60 minutesat room temperature with shaking. The plate was washed and read on anECL analyzer.

FIG. 8 provides a graphical representation of a curve prepared using theformat with mAb 3C2 as the detector antibody for the CK18 assay withrCK18fB. FIG. 9 provides a graphical representation of a curve preparedusing the format with mAb 3C2 as the detector antibody for the CK18assay with Apoptotic SK-BR3 Supernatant. The results clearly indicatedthat the format using mAb 3C2 as the capture antibody was more sensitivethan the format using mAb 3C2 as the detector antibody (See, e.g., FIGS.8 and 9), with at least a 0.5 log increase in sensitivity achieved.

The examples disclosed herein are merely illustrative and intended to benon-limiting. Moreover, it should be understood that various featuresand/or characteristics of differing embodiments herein may be combinedwith one another. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the scope of thepresent disclosure. Other embodiments will be apparent to those skilledin the art from consideration of the disclosure and practice of thevarious example embodiments disclosed herein. It is intended that thespecification and examples be considered as examples only, with the truescope of the invention being indicated by the claims.

It is also to be understood that, as used herein the terms “the,” “a,”or “an,” mean “at least one,” and should not be limited to “only one”unless explicitly indicated to the contrary. Unless otherwise indicated,all numbers used in the specification and claims are to be understood asbeing modified in all instances by the term “about,” whether or not sostated. It should also be understood that the precise numerical valuesused in the specification and claims form additional embodiments of theinvention, and are intended to include any ranges which can be narrowedto any two end points within the example ranges and values provided.Efforts have been made to ensure the accuracy of the numerical valuesdisclosed herein. Any measured numerical value, however, can inherentlycontain certain errors resulting from the standard deviation found inits respective measuring technique.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretyinto the specification to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Alsoincorporated by reference is any supplemental information that waspublished along with any of the aforementioned publications, patents andpatent applications. For example, some journal articles are publishedwith supplemental information that is typically available online.

What is claimed is:
 1. An antibody wherein the antibody has bindingspecificity to the amino acid sequence Ala Ser Ser Gly Leu Thr Val GluVal Asp (SEQ ID NO:1), wherein the antibody comprises a variable regionof the heavy chain amino acid sequence of SEQ ID NO:5, and a variableregion of the light chain amino acid sequence of SEQ ID NO:7.
 2. Theantibody of claim 1, wherein the antibody is coupled to a solid supportor a detection moiety.
 3. The antibody of claim 2, wherein the antibodyis directly bound to the solid support or the detection moiety.
 4. Theantibody of claim 2, wherein the antibody is indirectly bound to thesolid support or the detection moiety.
 5. The antibody of claim 2,wherein the detection moiety is selected from the group consisting ofelectrochemiluminescence labels or compounds, chemiluminescentcompounds, enzyme labels, fluorophores, chromogenic compounds,radiolabels, catalysts, colorimetric, labeled antibodies, a latexparticle, a magnetic particle, a paramagnetic particle, a radioactiveelement, fluorescent dyes, phosphorescent dyes, dye crystals, goldparticles, silver colloidal particles, selenium colloidal particles,metal chelates, coenzymes, electro active groups, oligonucleotides andstable radicals.
 6. The antibody of claim 5, wherein the metal chelateis a ruthenium, an osmium, or a europium metal chelate.
 7. The antibodyof claim 2, wherein the solid support is selected from the groupconsisting of a bead, a plate, a glass surface, a plastic surface, ametal surface, a polystyrene surface, a nitrocellulose surface, amicroparticle and a nanoparticle surface.
 8. The antibody of claim 7,wherein the solid support is a bead.